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US20250258162A1 - Regulatory t cell stability assay - Google Patents

Regulatory t cell stability assay

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US20250258162A1
US20250258162A1 US19/181,950 US202519181950A US2025258162A1 US 20250258162 A1 US20250258162 A1 US 20250258162A1 US 202519181950 A US202519181950 A US 202519181950A US 2025258162 A1 US2025258162 A1 US 2025258162A1
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ifn
cell population
cells
regulatory
tnf
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Yanbo Zhang
Devapregasan MOODLEY
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Abata Therapeutics Inc
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Abata Therapeutics Inc
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Assigned to ABATA THERAPEUTICS, INC. reassignment ABATA THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MOODLEY, Devapregasan, ZHANG, Yanbo
Assigned to ABATA THERAPEUTICS, INC. reassignment ABATA THERAPEUTICS, INC. CORRECTIVE ASSIGNMENT TO CORRECT THE ASSIGNEE ADDRESS PREVIOUSLY RECORDED AT REEL: 70987 FRAME: 644. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Assignors: MOODLY, DEVAPREGASAN, ZHANG, Yanbo
Publication of US20250258162A1 publication Critical patent/US20250258162A1/en
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Definitions

  • Regulatory T cells have potential for the treatment of diseases, such as autoimmune diseases, because they can target specific diseased cell types. Regulatory T cells can be useful, for example, in generating a local immune response that is specific to certain cell types and tissues associated with a disease of interest via an antigen-specific mechanism. Nonetheless, robust clinical scale and clinical grade manufacturing of stable antigen-specific regulatory T cells has been challenging. Thus, new methods of manufacturing, characterizing, culturing and expanding such regulatory T cells are needed.
  • a method of evaluating the stability profile of an isolated cell population comprising regulatory T cells comprises: (i) culturing the isolated cell population in the presence of two or more pro-inflammatory cytokines for a period of time; (ii) characterizing one or more markers of stability of the isolated cell population following the period of time; and (iii) evaluating the stability profile of the isolated cell population based on the characterizing of the one or more markers of stability.
  • Some aspects of the disclosure provide a method comprising (i) culturing an isolated cell population comprising regulatory T cells in the presence of two or more pro-inflammatory cytokines for a period of time; and (ii) characterizing one or more markers of stability of the isolated cell population following the period of time.
  • the two or more pro-inflammatory cytokines comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine cytokines selected from the group consisting of Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-12 (IL-12) (also known as IL-12p70), Tumor necrosis factor alpha (TNF- ⁇ ), Interleukin-17 (IL-17A), Interferon gamma (IFN- ⁇ ), Interferon beta (IFN- ⁇ ), Transforming growth factor beta 1 (TGF- ⁇ 1), and Interleukin-1 beta (IL-1 ⁇ ).
  • IL-2 Interleukin-2
  • IL-6 Interleukin-6
  • IL-12 also known as IL-12p70
  • IL-17A Interleukin-17
  • IFN- ⁇ Interferon gamma
  • IFN- ⁇ Interferon beta
  • TGF- ⁇ 1 Transforming growth factor beta 1
  • the two or more pro-inflammatory cytokines comprises IL-2 and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight cytokines selected from the group consisting of IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ .
  • the two or more pro-inflammatory cytokines comprises: (a) IL-2 and IL-6; (b) IL-2 and IL-12; (c) IL-2 and TNF- ⁇ ; (d) IL-2 and IL-17A; (e) IL-2 and IFN- ⁇ ; (f) IL-2 and IFN- ⁇ ; (g) IL-2 and TGF- ⁇ 1; or (h) IL-2 and IL-1 ⁇ .
  • the two or more pro-inflammatory cytokines comprises: (a) IL-2, IL-6, and at least one additional pro-inflammatory cytokine selected from: IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (b) IL-2, IL-12, and at least one additional pro-inflammatory cytokine selected from: IL-6, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (c) IL-2, TNF- ⁇ , and at least one additional pro-inflammatory cytokine selected from: IL-6, IL-12, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (d) IL-2, IL-17A, and at least one additional pro-inflammatory cytokine selected from: IL-6, IL-12, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL
  • the two or more pro-inflammatory cytokines comprises: (a) IL-2, IL-6, and at least two additional pro-inflammatory cytokines selected from: IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (b) IL-2, IL-12, and at least two additional pro-inflammatory cytokines selected from: IL-6, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (c) IL-2, TNF- ⁇ , and at least two additional pro-inflammatory cytokines selected from: IL-6, IL-12, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (d) IL-2, IL-17A, and at least two additional pro-inflammatory cytokines selected from: IL-6, IL-12, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL
  • the two or more pro-inflammatory cytokines comprises: (a) IL-2, IL-6, and at least three additional pro-inflammatory cytokines selected from: IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (b) IL-2, IL-12, and at least three additional pro-inflammatory cytokines selected from: IL-6, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (c) IL-2, TNF- ⁇ , and at least three additional pro-inflammatory cytokines selected from: IL-6, IL-12, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ ; (d) IL-2, IL-17A, and at least three additional pro-inflammatory cytokines selected from: IL-6, IL-12, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL
  • the two or more pro-inflammatory cytokines comprises IL-2, IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ .
  • the concentration of IL-2 is about 1000 U/mL and the concentration of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , TNF- ⁇ , IFN- ⁇ , and TGF- ⁇ 1 is about 1000 pg/mL.
  • the concentration of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , TNF- ⁇ , IFN- ⁇ , and TGF- ⁇ 1 is about 1000 pg/mL.
  • the two or more pro-inflammatory cytokines comprise IL-2, IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ .
  • the concentration of IL-2 is about 1000 U/mL and/or the concentration of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ is about 1000 pg/mL.
  • the concentration of IL-2 is about 1000 U/mL and the concentration of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ is about 1000 pg/mL.
  • the two or more pro-inflammatory cytokines further comprises Interleukin-22 (IL-22), Granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or Interleukin-23 (IL-23). In some embodiments, the two or more pro-inflammatory cytokines further comprises Interleukin-22 (IL-22), Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin-23 (IL-23).
  • IL-22 Interleukin-22
  • GM-CSF Granulocyte-macrophage colony-stimulating factor
  • IL-23 Interleukin-23
  • the concentration of any one of the pro-inflammatory cytokines is in the range of 0.01-10000 pg/mL, 0.01-1000 pg/mL, 0.01-100 pg/mL, 0.01-10 pg/mL, 0.01-1 pg/mL, 1-1000 pg/mL, 1-500 pg/mL, 1-100 pg/mL, 10-1000 pg/mL, 10-100 pg/mL, 50-500 pg/mL, 100-500 pg/mL, 100-750 pg/mL, 100-1000 pg/mL, 250-1000 pg/mL, 500-1000 pg/mL, 500-10000 pg/mL, 500-5000 pg/mL, 1000-10000
  • the concentration of IL-2 is in the range of 1-10000 U/mL, 1-7500 U/mL, 1-5000 U/mL, 1-2500 U/mL, 1-1000 U/mL, 100-5000 U/mL, 100-2500 U/mL, 100-1000 U/mL, 100-500 U/mL, 250-1000 U/mL, 250-2500 U/mL, 500-5000 U/mL, 500-2500 U/mL, 500-1000 U/mL, or 750-2000 U/mL.
  • the isolated cell population comprising regulatory T cells in the presence of two or more pro-inflammatory cytokines, as described herein, an anti-CD3antibody and an anti-CD28 antibody.
  • the anti-CD3 antibody and anti-CD28 antibody are attached to a solid support, e.g., a magnetic or polymeric bead.
  • the anti-CD3 antibody and anti-CD28 antibody are suspended in a matrix.
  • the period of time is at least 3, at least 6, at least 9, at least 12, at least 15, at least 20, or at least 25 days. In some embodiments, the period of time is 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days. The period of time may be 2-30 days, 3-25 days, 5-25 days, 5-20 days, 5-10 days, 10-30 days, 10-25 days, 10-20 days, 15-30 days, or 20-30 days. In some embodiments, the period of time is about 3 days. In some embodiments, the period of time is about 4 days. In some embodiments, the period of time is about 5 days.
  • the method further comprises characterizing the one or more markers of stability of the isolated cell population at regular intervals throughout the period of time.
  • the method comprises characterizing the one or more markers of stability of the isolated cell population every day, every second day, every third day, or every fourth day throughout the period of time.
  • the one or more markers of stability may be the biomarkers CD4, CD25, and/or FOXP3.
  • the biomarker of stability is CD4.
  • the biomarker of stability is CD25.
  • the biomarker of stability is FOXP3.
  • the one or more markers of stability comprise the methylation of a regulatory T cell-specific demethylation region (TSDR) at an endogenous FOXP3 locus and/or FOXP3 expression.
  • TSDR regulatory T cell-specific demethylation region
  • Some aspects of the disclosure provide a method of expanding a cell population comprising regulatory T cells.
  • the at least three additional pro-inflammatory cytokines are selected from the group consisting of Interleukin-6 (IL-6), Interleukin-12 (IL-12), Tumor necrosis factor (TNF- ⁇ ), Interleukin-17 (IL-17A), Interleukin-21 (IFN- ⁇ ), Interferon gamma (IFN- ⁇ ), Transforming growth factor beta 1 (TGF- ⁇ 1), and Interleukin-1b (IL-1 ⁇ ).
  • IL-6 Interleukin-6
  • IL-12 Interleukin-12
  • TNF- ⁇ Tumor necrosis factor
  • IL-17A Interleukin-17
  • IFN- ⁇ Interleukin-21
  • IFN- ⁇ Interferon gamma
  • TGF- ⁇ 1 Transforming growth factor beta 1
  • TGF- ⁇ 1 Interleukin-1b
  • the at least three additional pro-inflammatory cytokine comprises at least four, at least five, at least six, at least seven, or at least eight additional pro-inflammatory cytokines selected from the group consisting of Interleukin-6 (IL-6), Interleukin-12 (IL-12), Tumor necrosis factor (TNF- ⁇ ), Interleukin-17 (IL-17A), Interferon beta (IFN- ⁇ ), Interferon gamma (IFN- ⁇ ), Transforming growth factor beta 1 (TGF- ⁇ 1), and Interleukin-1b (IL-1 ⁇ ).
  • the at least three additional pro-inflammatory cytokines comprise IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ .
  • Some aspects of the disclosure provide a method of expanding a cell population comprising regulatory T cells comprising culturing an isolated cell population comprising regulatory T cells in the presence of Interferon beta (IFN- ⁇ ).
  • IFN- ⁇ Interferon beta
  • Some aspects of the disclosure provide a method of expanding a cell population comprising regulatory T cells comprising culturing an isolated cell population comprising regulatory T cells in the presence of Interferon gamma (IFN- ⁇ ).
  • IFN- ⁇ Interferon gamma
  • Some aspects of the disclosure provide a method of expanding a cell population comprising regulatory T cells comprising culturing an isolated cell population comprising regulatory T cells in the presence of Interleukin-1b (IL-1 ⁇ ).
  • IL-1 ⁇ Interleukin-1b
  • the concentration of any one of the pro-inflammatory cytokines is in the range of 0.01-10000 pg/mL, 0.01-10000 pg/mL, 0.01-1000 pg/mL, 0.01-100 pg/mL, 0.01-10 pg/mL, 0.01-1 pg/mL, 1-1000 pg/mL, 1-500 pg/mL, 1-100 pg/mL, 10-1000 pg/mL, 10-100 pg/mL, 50-500 pg/mL, 100-500 pg/mL, 100-750 pg/mL, 100-1000 pg/mL, 250-1000 pg/mL, 500-1000 pg/mL, 500-10000 pg/mL, 500-5000
  • the concentration of IL-2 is in the range of 1-10000 U/mL, 1-7500 U/mL, 1-5000 U/mL, 1-2500 U/mL, 1-1000 U/mL, 100-5000 U/mL, 100-2500 U/mL, 100-1000 U/mL, 100-500 U/mL, 250-1000 U/mL, 250-2500 U/mL, 500-5000 U/mL, 500-2500 U/mL, 500-1000 U/mL, or 750-2000 U/mL, optionally wherein the concentration of IL-2 is 1000 U/mL.
  • the concentration of each of the at least three additional pro-inflammatory cytokines is in the range of 100-1000 pg/mL.
  • the method further comprises characterizing the relative abundance of IFN- ⁇ + cells within the cell population after (i).
  • at least 70% of the cells of the isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at an endogenous FOXP3 locus and/or at least 80% of the cells of the isolated cell population are FOXP3+.
  • At least 70% of the cells of the isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at an endogenous FOXP3 locus. In some embodiments, prior to the culturing of step (i), at least 80% of the cells of the isolated cell population are FOXP3 + . In some embodiments, prior to the culturing of step (i), at least 70% of the cells of the isolated cell population are stable regulatory T cells comprising a hypomethylated TSDR at an endogenous FOXP3 locus and at least 80% of the cells of the isolated cell population are FOXP3 + .
  • culturing the isolated cell population comprises culturing the isolated cell population in a cell media. In some embodiments, culturing the isolated cell population comprises replacing the cell media with new cell media at regular intervals.
  • the cell media may comprise a buffer (e.g., phosphate-buffered saline (PBS)), bovine serum albumin, human serum albumin, and/or EDTA.
  • PBS phosphate-buffered saline
  • bovine serum albumin bovine serum albumin
  • human serum albumin and/or EDTA.
  • the isolated cell population is cultured in the presence of one or more agents that activate T cells within the isolated cell population.
  • the one or more agents that activate T cells are antigen-presenting cells or agents that mimic antigen-presenting cells.
  • the agents that mimic antigen-presenting cells may be three-dimensional beads. In some embodiments, the three-dimensional beads are attached to activation signals. In some embodiments, the agents that mimic antigen-presenting cells are three-dimensional beads that are attached to anti-CD3, anti-CD28, and/or anti-CD137 molecules. In some embodiments, the three-dimensional beads are attached to anti-CD3 molecules. In some embodiments, the three-dimensional beads are attached to anti-CD28 molecules. In some embodiments, the three-dimensional beads are attached to anti-CD137 molecules.
  • the agents are polystyrene beads coated with a mixture of monoclonal antibodies against the CD3 and CD28 cell surface molecules of human T cells.
  • the one or more agents that activate T cells are phorbol 12-myristate 13-acetate (PMA) and ionomycin.
  • culturing the isolated cell population comprises replacing the cell media with new cell media every 2-5 days, optionally every 3-4 days.
  • the isolated cell population is a stable cell population if at least 80% of the regulatory T cells comprise a hypomethylated TSDR and an endogenous FOXP3 locus and/or at least 80% of the cells of the isolated cell population are FOXP3 + . In some embodiments, the isolated cell population is a stable cell population if at least 80% of the regulatory T cells comprise a hypomethylated TSDR and an endogenous FOXP3 locus. In some embodiments, the isolated cell population is a stable cell population if at least 80% of the cells of the isolated cell population are FOXP3+.
  • the isolated cell population is a stable cell population if at least 80% of the regulatory T cells comprise a hypomethylated TSDR and an endogenous FOXP3 locus and at least 80% of the cells of the isolated cell population are FOXP3+.
  • FIG. 1 provides a schematic of an exemplary method of a regulatory T cell stability assay of the disclosure.
  • FIG. 2 provides an exemplary regulatory T cell gating strategy used in the regulatory T cell stability assay of the disclosure that utilizes fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • FIG. 3 provides graphs showing that regulatory T cells are expanded in the presence of pro-inflammatory cytokines.
  • FIGS. 5 A- 5 B provide graphs showing the expansion and purity of a cell population comprising regulatory T cells following culture and expansion in the presence of pro-inflammatory cytokines for up to 23 days.
  • FIG. 6 provides a graph showing the ability of an isolated cell population comprising regulatory T cells to expand when cultured in the presence of IL-2 and TNF- ⁇ .
  • FIGS. 7 A- 7 D provide graphs showing the percentage of CD4 + Foxp3 + cells and percentage of TSDR demethylation in stable regulatory T cells following culture and expansion in the presence of IL-2 or two different mixtures of pro-inflammatory cytokines for three days. Data for three different runs are shown.
  • FIGS. 8 A- 8 B is a graph showing the percentage of CD4 + Foxp3 + cells and percentage of TSDR demethylation in conventional T cells following culture and expansion in the presence of IL-2 or two different mixtures of pro-inflammatory cytokines for three days.
  • FIG. 9 A is a graph showing that FOXP3 expression on Tregs remained stable over a 5-day culture period, even in the presence of the pro-inflammatory Mix-6.
  • In vitro induced Tregs iTregs
  • FIG. 9 B is a graph showing that the demethylation of the TSDR region remained stable and high on Tregs even in the presence of the pro-inflammatory Mix-6; TSDR demethylation remained below 2% for iTregs over time under different culture conditions.
  • FIG. 9 C and FIG. 9 D are graphs showing that Tregs maintained low IL-2 ( FIG. 9 C ) and IFN- ⁇ ( FIG.
  • FIGS. 10 A- 10 C provide graphs showing the percentage TSDR demethylation in conventional T cells ( FIG. 10 A ), stable regulatory T cells ( FIG. 10 B ), and stable regulatory T cells engineered to express an exogenous TCR ( FIG. 10 C ) following culture and expansion in the presence of IL-2 or two different mixtures of pro-inflammatory cytokines for five days.
  • FIGS. 11 A- 11 C provide graphs showing the percentage of CD4 + Foxp3 + cells in conventional T cells ( FIG. 11 A ), stable regulatory T cells ( FIG. 11 B ), and stable regulatory T cells engineered to express an exogenous TCR ( FIG. 11 C ) following culture and expansion in the presence of IL-2 or two different mixtures of pro-inflammatory cytokines for five days.
  • FIGS. 12 A- 12 C provide graphs showing the percentage of IFN- ⁇ + cells in conventional T cells ( FIG. 12 A ), stable regulatory T cells ( FIG. 12 B ), and stable regulatory T cells engineered to express an exogenous TCR ( FIG. 12 C ) following culture and expansion in the presence of IL-2 or two different mixtures of pro-inflammatory cytokines for five days.
  • the present disclosure provides, in some aspects, methods and compositions for the assessment of regulatory T cell stability over time under inflammatory conditions.
  • the present disclosure provides methods and compositions for expanding regulatory T cells in culture.
  • Cell populations comprising regulatory T cells e.g., engineered regulatory T cells comprising an antigen-specific T cell receptor (TCR)
  • TCR antigen-specific T cell receptor
  • such cell populations comprise stable (e.g., terminally differentiated) regulatory T cells that are capable of persisting in vivo for extended periods and may provide therapeutic benefit for months or years following a single dose (or, in some instances, multiple doses).
  • These stable regulatory T cells are also, in some embodiments, able to maintain their Treg phenotype in the presence of pro-inflammatory triggers (e.g., pro-inflammatory cytokines).
  • pro-inflammatory triggers e.g., pro-inflammatory cytokines
  • regulatory T cells are also referred to as “Tregs”.
  • Tregs regulatory T cells
  • pro-inflammatory conditions e.g., pro-inflammatory conditions that approximate the in vivo conditions these cells may experience within a human body.
  • pro-inflammatory conditions e.g., pro-inflammatory conditions that approximate the in vivo conditions these cells may experience within a human body.
  • the assay presented herein is an ex vivo assay that assesses whether Tregs will maintain their stable Treg phenotype once exposed to inflammatory conditions, and thereby, whether the Treg are safe for administration to a subject.
  • a method of evaluating the stability profile of an isolated cell population comprising regulatory T cells comprises culturing the isolated cell population in the presence of one or more (e.g., two or more) pro-inflammatory cytokines (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 pro-inflammatory cytokines) for a period of time, characterizing one or more markers of stability (e.g., FOXP3 expression or the methylation status of a regulatory T cell-specific demethylation region (TSDR) at an endogenous FOXP3 locus) of the isolated cell population following the period of time, and evaluating the stability profile of the isolated cell population based on the characterizing of the one or more markers of stability.
  • pro-inflammatory cytokines e.g., 2, 3, 4, 5, 6, 7, 8, or 9 pro-inflammatory cytokines
  • markers of stability e.g., FOXP3 expression or the methylation status of a regulatory T cell-specific demethylation region (TSDR) at an endogenous FOXP3 locus
  • the method further comprises determining the levels of pro-inflammatory cytokines produced by the cells (e.g., regulatory T cells) of the isolated cell population. In some embodiments, the method further comprises determining the levels of intracellular IFN- ⁇ produced by the cells (e.g., regulatory T cells) of the isolated cell population. Specifically, the method, in some embodiments, involves a determination of the relative abundance of IFN- ⁇ +cells within the isolated cell population after exposure to inflammatory cytokines. In some embodiments, the method further comprises determining the levels of intracellular IL-17 produced by the cells (e.g., regulatory T cells) of the isolated cell population.
  • the method involves a determination of the relative abundance of IL-17 + cells within the isolated cell population after exposure to inflammatory cytokines. In some embodiments, the method further comprises determining the levels of intracellular TNF- ⁇ produced by the cells (e.g., regulatory T cells) of the isolated cell population. In some embodiments, the method involves a determination of the relative abundance of TNF- ⁇ + cells within the isolated cell population after exposure to inflammatory cytokines. In some embodiments, the method further comprises determining the levels of IL-2 produced by the cells (e.g., regulatory T cells) of the isolated cell population. Accordingly, in some embodiments, involves a determination of the relative abundance of IL-2 producing cells within the isolated cell population after exposure to inflammatory cytokines.
  • This characterization is intended to determine whether the regulatory T cells of the isolated cell population adopt effector cell function in response to inflammatory stimuli. Furthermore, large-scale production of these regulatory T cells necessitates, in some embodiments, efficient methods of culturing and expanding regulatory T cells. Surprisingly, the data provided herein shows that culturing regulatory T cells in the presence of certain pro-inflammatory cytokines and specific combinations of pro-inflammatory cytokines facilitate high levels of regulatory T cell expansion.
  • a regulatory T cell also referred to as a “Treg” is a T cell that modulates the immune system. Regulatory T cells are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Regulatory T cells are thought to be derived from the same lineage as na ⁇ ve CD4 + cells and express the biomarkers CD4, CD25, and FOXP3.
  • the regulatory T cells e.g., stable regulatory T cells
  • TCR human T cell receptor
  • a stable, or thymic, regulatory T cell is a regulatory T cell that comprises a hypomethylated TSDR at an endogenous FOXP3 locus and expresses CD4, CD25, and FOXP3.
  • a regulatory T cell is considered “stable” if it is terminally differentiated, i.e., it has lost its ability to change its cell fate.
  • the genome organizer SATB1 (special AT-rich sequence-binding protein) binds to specific genomic sites from the CD4 + CD8 + thymocyte stage to open up the chromatin and activate super-enhancers associated with many regulatory T cell signature genes such as FOXP3, IL2RA, (CD25), CTLA4, IKZF2 (HELIOS), and IFZF4 (EOS).
  • SATB1 and MLL4 myeloid/lymphoid or mixed-lineage leukemia 4
  • an enzyme involved in enhancer priming commonly occupy the newly identified conserved enhancer region, designated conserved noncoding sequence 0 (CNS0), at the FOXP3 locus, with subsequent activation of the enhancers at CNS3 and CNS2, and then the promoter.
  • CNS0 conserved noncoding sequence 0
  • TSDR Hypomethylation of the TSDR, which is an evolutionary conserved CpG-rich regulatory element of the FOXP3 gene, is associated with expression of FOXP3.
  • a TSDR of an endogenous FOXP3 locus is hypomethylated when the methyl group from one or more methylated cytosines in the TSDR have been removed to replace the methylated cytosine(s) with cytosine.
  • measurement of the methylation status of the TSDR of a FOXP3 locus is as described in Kressler et. al.
  • NGS next-generation sequencing
  • a stable regulatory T cell comprises a hypomethylated TSDR at an endogenous FOXP3 locus in the presence of pro-inflammatory conditions for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, or 25 days.
  • a stable regulatory T cell comprises a hypomethylated TSDR at an endogenous FOXP3 locus and expresses CD 4 , CD 25 , and FOXP3 in the presence of pro-inflammatory conditions for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, or 25 days.
  • a stable regulatory T cell has a low level of intracellular IFN ⁇ expression.
  • a stable regulatory T cell maintains a low level of intracellular IFN ⁇ expression in the presence of pro-inflammatory conditions for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, or 25 days.
  • a stable regulatory T cell does not adopt or have effector cell function.
  • the one or more markers of stability of a regulatory T cell are one or more biomarkers that are representative of a stable regulatory T cell.
  • a marker of stability of a regulatory T cell is a hypomethylated TSDR at an endogenous FOXP3 locus.
  • a marker of stability of a regulatory T cell is CD4, CD25, or FOXP3.
  • a marker of stability (e.g., CD4, CD25, or FOXP3) is characterized by determining expression or activity of said marker.
  • a regulatory T cell retains a marker of stability if the regulatory T cell maintains a hypomethylated TSDR at an endogenous FOXP3 locus after a period of time.
  • a regulatory T cell retains a marker of stability if the regulatory T cell maintains expression of CD4, CD25, and/or FOXP3 after a period of time. In some embodiments, a regulatory T cell retains a marker of stability if the regulatory T cell maintains high levels of expression of CD4, CD25, and/or FOXP3 after a period of time.
  • a stable regulatory T cell further exhibits one or more of the following functions: (i) cytokine secretion activity (e.g., secretion of IL-10, IL-4, IL-6, IL-11, and/or IL-13) when activated (e.g., activated by anti-CD3 and anti-CD28 antibodies); (ii) expression of activation markers associated with regulatory T cells (e.g., expression of CD69, 4-1BB, CD25, CD71, and/or CTLA-4) when activated (e.g., activated by anti-CD3 and anti-CD28 antibodies); and/or (iii) suppression activity (e.g., the ability of the stable regulatory T cell to suppress the activation of conventional T cells having specificity towards a shared target peptide) when activated (e.g., activated by anti-CD3 and anti-CD28 antibodies).
  • cytokine secretion activity e.g., secretion of IL-10, IL-4, IL-6, IL-11, and/
  • a subpopulation of those cells may express higher levels of CD25 relative to another subpopulation of CD25 + cells in that population (or relative to all the other cells CD25 + cells in the population).
  • the former subpopulation would be considered to be CD25 high
  • the latter subpopulation would be considered only CD25 + (not CD25 high ).
  • a regulatory T cell (e.g., a stable regulatory T cell) is CD4 + .
  • a regulatory T cell (e.g., a stable regulatory T cell) is CD127 ⁇ /lo .
  • CD127 ⁇ /lo means that the cell is either CD127 ⁇ or CD127 lo .
  • a CD127 ⁇ cell is a cell that does not express CD127, does not express a detectable level of CD127 (e.g., by FACS), or expresses CD127 below a threshold or control level.
  • a regulatory T cell (e.g., a stable regulatory T cell) is FOXP3 + .
  • a regulatory T cell (e.g., a stable regulatory T cell) is a CD25 +/high CD4 + CD127 ⁇ /lo cell. That is, the regulatory T cell expresses, or expresses a high level of CD25, expresses CD4, and does not express, or expresses a low level of, CD127. In some embodiments, a regulatory T cell does not express CD127. In some embodiments, a regulatory T cell (e.g., a stable regulatory T cell) is a CD25 +/high CD4 + CD127 ⁇ /10/ CD45RA + cell.
  • the regulatory T cells expresses, or expresses a high level of CD25, expresses CD4 and CD45RA, and does not express, or expresses a low level of, CD127.
  • a regulatory T cell expresses FOXP3.
  • a regulatory T cell is a CD25 +/high CD4+CD127 ⁇ /lo FOXP3 + cell.
  • the regulatory T cells expresses, or expresses a high level of CD25, expresses CD4 and FOXP3, and does not express, or expresses a low level of, CD127.
  • the control level of a biomarker in a regulatory T cell is the expression level of the biomarker (e.g., CD127) in a conventional T cell or a CD8 + T cell.
  • a regulatory T cell expresses a low level of CD127 if its expression of CD127 is lower than (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than) expression of CD127 in a conventional T cell.
  • a stable regulatory T cell of an isolated cell population maintains a hypomethylated TSDR at an endogenous FOXP3 locus (e.g., in the presence of one or more pro-inflammatory cytokines) over time (i.e., the TSDR at the endogenous FOXP3 locus of viable cells (e.g., in culture, in a cryoprotective agent, and/or in vivo) is hypomethylated for a certain measurable period of time).
  • a stable regulatory T cell of an isolated cell population may maintain a hypomethylated TSDR at an endogenous FOXP3 locus (e.g., in the presence of one or more pro-inflammatory cytokines) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days (e.g., in culture, in a cryoprotective agent, and/or in vivo after being obtained from a subject or following transduction with a nucleic acid expressing a TCR).
  • an endogenous FOXP3 locus e.g., in the presence of one or more pro-inflammatory cytokines
  • a stable regulatory T cell maintains a hypomethylated TSDR at an endogenous FOXP3 locus (e.g., in the presence of one or more pro-inflammatory cytokines) for more than 5 days, more than 10 days, more than 15 days, or more than 20 days (e.g., in culture, in a cryoprotective agent, and/or in vivo after being obtained from a subject or following transduction with a nucleic acid expressing a TCR).
  • an endogenous FOXP3 locus e.g., in the presence of one or more pro-inflammatory cytokines
  • a stable regulatory T cell maintains a hypomethylated TSDR at an endogenous FOXP3 locus (e.g., in the presence of one or more pro-inflammatory cytokines) for 1-20 days, 1-10 days, 1-5 days, 5 -30 days, 5-20 days, 10-40days, or 25-50 days (e.g., in culture, in a cryoprotective agent, and/or in vivo after being obtained from a subject or following transduction with a nucleic acid expressing a TCR).
  • an endogenous FOXP3 locus e.g., in the presence of one or more pro-inflammatory cytokines for 1-20 days, 1-10 days, 1-5 days, 5 -30 days, 5-20 days, 10-40days, or 25-50 days (e.g., in culture, in a cryoprotective agent, and/or in vivo after being obtained from a subject or following transduction with a nucleic acid expressing a TCR).
  • a regulatory T cell of an isolated cell population of the disclosure in some embodiments, is an autologous cell.
  • autologous in this context refers to cells that have been obtained from the same subject to which they are subsequently administered (e.g., following engineering to introduce an exogenous TCR into the cells).
  • a population of cells may be obtained from a subject, subjected to the methods described herein, and then administered to the same subject (from which the population of cells was originally obtained) to treat an autoimmune disease.
  • the population of cells administered to the subject comprise autologous regulatory T cells.
  • a regulatory T cell of the disclosure in some embodiments, is an allogenic cell.
  • the term allogenic in this context refers to cells that have been obtained from one subject and then administered to another subject.
  • a population of cells may be obtained from a subject, subjected to the methods described herein, and then administered to another subject in order to treat an autoimmune disease.
  • regulatory T cells are obtained from a subject diagnosed with, or suspected of having, an autoimmune disease.
  • the autoimmune disease may be selected from Multiple Sclerosis (e.g., progressive Multiple Sclerosis), Type 1 Diabetes, and Inclusion Body Myositis.
  • the autoimmune disease is progressive Multiple Sclerosis (e.g., progressive Multiple Sclerosis).
  • regulatory T cells are obtained from a subject diagnosed with or suspected of having Multiple Sclerosis (e.g., progressive Multiple Sclerosis).
  • the autoimmune disease is Type 1 Diabetes.
  • regulatory T cells are obtained from a subject diagnosed with or suspected of having Type 1 Diabetes.
  • the autoimmune disease is Inclusion Body Myositis.
  • regulatory T cells are obtained from a subject diagnosed with or suspected of having Inclusion Body Myositis.
  • the stability profile of regulatory T cells is a valuable tool in characterizing the quality of an isolated cell population comprising regulatory T cells, particularly if the isolated cell population is intended to be used to therapeutic purposes.
  • the stability profile of regulatory T cells may be evaluated by subjecting the regulatory T cells to pro-inflammatory conditions (e.g., such as those that might be experienced within a patient).
  • the stability profile of regulatory T cells may include an evaluation of the methylation status of a TSDR at an endogenous FOXP3 locus, the expression and/or activity of CD4, CD25, and/or FOXP3.
  • the stability profile of an isolated cell population comprising regulatory T cells is evaluated by (i) culturing the isolated cell population in the presence of two or more pro-inflammatory cytokines for a period of time; (ii) characterizing one or more markers of stability of the isolated cell population following the period of time; and (iii) evaluating the stability profile of the isolated cell population based on the characterizing of the one or more markers of stability.
  • a method of evaluating the stability profile of a regulatory T cell comprises culturing an isolated cell population comprising the regulatory T cell in the presence of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine pro-inflammatory cytokines.
  • a method of evaluating the stability profile of a regulatory T cell comprises culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, TGF- ⁇ 1, and at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) additional pro-inflammatory cytokine selected from: IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , and IL-1 ⁇ .
  • a method of evaluating the stability profile of a regulatory T cell comprises culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ .
  • the isolated cell population is cultured in the presence of two or more pro-inflammatory cytokines under activating conditions. In some embodiments, the isolated cell population is cultured in the presence of two or more pro-inflammatory cytokines and anti-CD3 and anti-CD28 antibodies.
  • the anti-CD3 and anti-CD8 antibodies are attached to a magnetic bead, such as CD3/CD28 DynabeadsTM.
  • CD3/CD28 DynabeadsTM are uniform 4.5 ⁇ m, super-paramagnetic polystyrene beads coated with a mixture of monoclonal antibodies against the CD3 and CD28 cell surface molecules of human T cells.
  • the anti-CD3 and anti-CD8 antibodies are suspended in a matrix, such as TransactTM.
  • a matrix such as TransactTM.
  • the regulatory T cells express an exogenous TCR
  • the isolated cell population is cultured in the presence of two or more pro-inflammatory cytokines and a TCR ligand, e.g., a peptide MHC complex.
  • the peptide MHC complex is presented by an antigen presenting cell.
  • the peptide-MHC complex is presented by a multimer reagent, e.g., a tetramer or dextramer.
  • the isolated cell population is cultured in the presence of one or more pro-inflammatory cytokines, phorbol 12-myristate 13-acetate (PMA) and ionomycin to activate intracellular signaling pathways.
  • PMA and ionomycin function to activate the T cells of the isolated cell population through an activation of protein kinase C (PKC) and nuclear factor of activated T-cell (NFAT) signaling pathways.
  • PKC protein kinase C
  • NFAT nuclear factor of activated T-cell
  • the isolated cell population is activated after exposing the isolated cell population to pro-inflammatory conditions (e.g., culturing in the presence of one or more pro-inflammatory cytokines for a period of time).
  • a method of evaluating the stability profile of a regulatory T cell comprises culturing an isolated cell population comprising the regulatory T cell in the presence of pro-inflammatory cytokines for a period of time.
  • a period of time in relation to the length of time for culturing an isolated population comprising regulatory T cells may be as short as 1, 2, or 3 days, or as long as 2 or 3 months.
  • a period of time is about or at least 3, at least 4, at least 5 at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 15, at least 20, at least 23, at least 25, or at least 30 days.
  • Cells may be plated in a cell culture dish or a multiwell plate (e.g., 96-well plate) at a concentration of at least 10 2 cells/mL, at least 10 3 cells/mL, at least 10 4 cells/mL, at least 10 5 cells/mL, at least 10 6 cells/mL, at least 10 7 cells/mL, at least 10 8 cells/mL, at least 10 9 cells/mL, or at least 10 10 cells/mL.
  • a multiwell plate e.g., 96-well plate
  • Characterizing the one or more markers of stability may involve, in some embodiments, determining the expression and/or activity of the one or more markers of stability.
  • characterizing CD4 may involve determining the expression and/or activity of CD4.
  • characterizing CD25 may involve determining the expression and/or activity of CD25.
  • characterizing FOXP3 may involve determining the expression and/or activity of FOXP3.
  • antibody-based assays include, but are not limited to immunohistochemistry, immunohistochemical staining and enzyme-linked immunosorbance assay (ELISA).
  • ELISA is a quantitative assay that may be used to detect secreted cytokines at the protein level.
  • FACS-based assays include but are not limited to flow cytometry (FCM).
  • Cytokine bead arrays may be used to detect a panel of cytokines in a multiplex fashion using small sample volumes.
  • cytokine bead array see Amsen D, et al. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5. PMID: 19347295; PMCID: PMC2698024.
  • kits such as LEGENDPLEXTM (BioLegend, Inc.). Examples of measuring RNA or DNA include, but are not limited to, real-time quantitative polymerase chain reaction (Q-PCR), Real-Time PCR.
  • a method of culturing an isolated cell population comprising regulatory T cells involves culturing the cell population in the presence of TNF- ⁇ . In some embodiments, a method of culturing an isolated cell population comprising regulatory T cells involves culturing the cell population in the presence of IL-2 and TNF- ⁇ . In some embodiments, culturing an isolated cell population comprising regulatory T cells in the presence of TNF- ⁇ (e.g., IL-2 and TNF- ⁇ ) results in an expanded cell population (e.g., an expanded cell population having higher abundance of regulatory T cells relative to a control, e.g., an isolated cell population cultured in the absence of TNF- ⁇ ).
  • TNF- ⁇ e.g., IL-2 and TNF- ⁇
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, IL-6, and at least two (e.g., 2, 3, 4, 5, 6, or 7) additional pro-inflammatory cytokines selected from: IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ .
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, TNF- ⁇ , and at least two (e.g., 2, 3, 4, 5, 6, or 7) additional pro-inflammatory cytokines selected from: IL-6, IL-12, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ .
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, IFN- ⁇ , and at least two (e.g., 2, 3, 4, 5, 6, or 7) additional pro-inflammatory cytokines selected from: IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ .
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, IFN- ⁇ , and at least two (e.g., 2, 3, 4, 5, 6, or 7) additional pro-inflammatory cytokines selected from: IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , TGF- ⁇ 1, and IL-1 ⁇ .
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, TGF- ⁇ 1, and at least two (e.g., 2, 3, 4, 5, 6, or 7) additional pro-inflammatory cytokines selected from: IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , and IL-1 ⁇ .
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, IL-1 ⁇ , and at least two (e.g., 2, 3, 4, 5, 6, or 7) additional pro-inflammatory cytokines selected from: IL-6, IL-12, TNF- ⁇ , IL-17A, IFN- ⁇ , IFN- ⁇ , and TGF- ⁇ 1.
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , TNF- ⁇ , IFN- ⁇ , and TGF- ⁇ 1.
  • a method of culturing an isolated cell population comprising the regulatory T cell in the presence of IL-2, IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ .
  • the concentration of any one of the pro-inflammatory cytokines may be in the range of 0.01-10000 pg/mL, 0.01-10000 pg/mL, 0.01-1000 pg/mL, 0.01-100 pg/mL, 0.01-10 pg/mL, 0.01-1 pg/mL, 1-1000 pg/mL, 1-500 pg/mL, 1-100 pg/mL, 10-1000 pg/mL, 10-100 pg/mL, 50-500 pg/mL, 100-500 pg/mL, 100-750 pg/mL, 100-1000 pg/mL, 250-1000 pg/mL, 500-1000 pg/mL, 500-10000 pg/mL, 500-5000 pg/
  • the concentration of IL-2 is in the range of 1-10000 U/mL, 1-7500 U/mL, 1-5000 U/mL, 1-2500 U/mL, 1-1000 U/mL, 100-5000 U/mL, 100-2500 U/mL, 100-1000 U/mL, 100-500 U/mL, 250-1000 U/mL, 250-2500 U/mL, 500-5000 U/mL, 500-2500 U/mL, 500-1000 U/mL, or 750-2000 U/mL. In some embodiments, the concentration of IL-2 is about 500, about 1000, about 1500, or about 2000 U/ml.
  • the concentration of IL-2 is about 1000 U/mL and the concentration of TNF- ⁇ is about 1000 pg/mL. In some embodiments, the concentration of IL-2 is about 100-2000 U/mL and the concentration of IL-17A is about 100-2000 pg/mL. In some embodiments, the concentration of IL-2 is about 500-1500 U/mL and the concentration of IL-17A is about 500-1500 pg/mL. In some embodiments, the concentration of IL-2 is about 1000 U/mL and the concentration of IL-17A is about 1000 pg/mL. In some embodiments, the concentration of IL-2 is about 100-2000 U/mL and the concentration of IFN- ⁇ is about 100-2000 pg/mL.
  • Culturing the isolated cell population may involve culturing the isolated cell population in a cell media.
  • the cell media may be replaced and/or supplemented with new or fresh cell media at regular intervals.
  • the cell media may be replaced and/or supplemented with new or fresh cell media every day, every second day, every third day, every fourth day, every fifth day, every six days, or once per week.
  • the cell media may be replaced and/or supplemented with new or fresh cell media every 1-5 days, 1-4 days, 1-3 days, 1-2 days, 2-5 days, 2-4 days, 2-3 days, 3-5 days, or 3-4 days.
  • an isolated cell population comprising regulatory T cells may be a modified version of the culture conditions may be found in Chakraborty R, et al. Robust and cost - effective expansion of human regulatory T cells highly functional in a xenograft model of graft - versus - host disease. Haematologica. 2013 April;98(4):533-7. doi: 10.3324/haematol.2012.076430. Epub 2012 Dec. 14. PMID: 23242592; PMCID: PMC3659983 or Siemasko, K. In Vitro Expanded CD 4+ CD 25+ Foxp 3+ Regulatory T Cells Maintain a Normal Phenotype and Suppress Immune - Mediated Ocular Surface Inflammation. Invest. Ophthalmol. Vis. Sci. 2008; 49(12):5434-5440. doi: doi.org/10.1167/iovs.08-2075.
  • the two or more pro-inflammatory cytokines comprise IL-2, IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ , optionally wherein the concentration of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ is about 1000 pg/mL, optionally wherein the concentration of IL-2 is 1000 U/mL.
  • the one or more markers of stability comprise the biomarkers CD4, CD25, and/or FOXP3.
  • the one or more markers of stability comprise the methylation of a regulatory T cell-specific demethylation region (TSDR) at an endogenous FOXP3 locus and/or FOXP3 expression.
  • TSDR regulatory T cell-specific demethylation region
  • culturing the isolated cell population comprises culturing the isolated cell population in a cell media.
  • culturing the isolated cell population comprises replacing the cell media with new cell media at regular intervals.
  • the cell media comprises phosphate-buffered saline (PBS), bovine serum albumin, human serum albumin, and/or EDTA.
  • PBS phosphate-buffered saline
  • bovine serum albumin bovine serum albumin
  • human serum albumin and/or EDTA.
  • culturing the isolated cell population comprises replacing the cell media with new cell media every 2-5 days, optionally every 3-4 days.
  • the isolated cell population is a stable cell population if at least 80% of the regulatory T cells comprise a hypomethylated TSDR and an endogenous FOXP3 locus and/or at least 80% of the cells of the isolated cell population are FOXP3+.
  • a method of expanding a cell population comprising culturing an isolated cell population comprising regulatory T cells in the presence of IL-2 and at least three additional pro-inflammatory cytokines.
  • the at least three additional pro-inflammatory cytokines are selected from the group consisting of Interleukin-6 (IL-6), Interleukin-12 (IL-12), Tumor necrosis factor (TNF- ⁇ ), Interleukin-17 (IL-17A), Interleukin-21 (IFN- ⁇ ), Interferon gamma (IFN- ⁇ ), Transforming growth factor beta 1 (TGF- ⁇ 1), and Interleukin-1b (IL-1 ⁇ ).
  • the at least three additional pro-inflammatory cytokine comprises at least four, at least five, at least six, at least seven, or at least eight additional pro-inflammatory cytokines selected from the group consisting of Interleukin-6 (IL-6), Interleukin-12 (IL-12), Tumor necrosis factor (TNF- ⁇ ), Interleukin-17 (IL-17A), Interferon beta (IFN- ⁇ ), Interferon gamma (IFN- ⁇ ), Transforming growth factor beta 1 (TGF- ⁇ 1), and Interleukin-1b (IL-1 ⁇ ).
  • IL-6 Interleukin-6
  • IL-12 Interleukin-12
  • TNF- ⁇ Tumor necrosis factor
  • IFN- ⁇ Interleukin-17
  • IFN- ⁇ Interferon beta
  • IFN- ⁇ Interferon gamma
  • TGF- ⁇ 1 Transforming growth factor beta 1
  • TGF- ⁇ 1 Interleukin-1b
  • a method of expanding a cell population comprising culturing an isolated cell population comprising regulatory T cells in the presence of Interleukin-17 (IL-17A).
  • IL-17A Interleukin-17
  • a method of expanding a cell population comprising culturing an isolated cell population comprising regulatory T cells in the presence of Interferon beta (IFN- ⁇ ).
  • IFN- ⁇ Interferon beta
  • a method of expanding a cell population comprising culturing an isolated cell population comprising regulatory T cells in the presence of Interleukin-1b (IL-1 ⁇ ).
  • IL-1 ⁇ Interleukin-1b
  • a method of expanding a cell population comprising culturing an isolated cell population comprising regulatory T cells in the presence of Tumor necrosis factor alpha (TNF- ⁇ ) and IL-2.
  • TNF- ⁇ Tumor necrosis factor alpha
  • IL-2 Tumor necrosis factor alpha
  • the one or more additional pro-inflammatory cytokines is Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-12 (IL-12), Tumor necrosis factor alpha (TNF- ⁇ ), Interleukin-17 (IL-17A), Interferon gamma (IFN- ⁇ ), Interferon beta (IFN- ⁇ ), Transforming growth factor beta 1 (TGF- ⁇ 1), and Interleukin-1 beta (IL-1 ⁇ ).
  • any one of the pro-inflammatory cytokines is in the range of 0.01-10000 pg/mL, 0.01-10000 pg/mL, 0.01-1000 pg/mL, 0.01-100 pg/mL, 0.01-10 pg/mL, 0.01-1 pg/mL, 1-1000 pg/mL, 1-500 pg/mL, 1-100 pg/mL, 10-1000 pg/mL, 10-100 pg/mL, 50-500 pg/mL, 100-500 pg/mL, 100-750 pg/mL, 100-1000 pg/mL, 250-1000 pg/mL, 500-1000 pg/mL, 500-10000 pg/mL, 500-5000 pg/mL, 1000-10000 pg/mL, 1000-5000 pg/mL, or 100-10000 pg/mL.
  • culturing the isolated cell population comprises culturing the isolated cell population in a cell media.
  • culturing the isolated cell population comprises replacing the cell media with new cell media at regular intervals.
  • culturing the isolated cell population comprises replacing the cell media with new cell media every 2-5 days, optionally every 3-4 days.
  • FIG. 1 provides a schematic of a regulatory T cell stability assay of the disclosure, described below.
  • CD4 + CD25 + CD127 low regulatory T cells were first isolated from PBMCs from a healthy donor using immunomagnetic cell isolation kit (StemCell Technologies, Cat#18063). Flow cytometry was then used to sort for CD4 + CD25 + CD127 low CD45RA+ Tregs (FACS). Sorted regulatory T cells were expanded for 8 days in the presence of human T-activator CD3/CD28 DynabeadsTM (Life Technologies, Cat#11132D), cell culture medium, and human recombinant IL-2. Post expansion, the regulatory T cell population was characterized using flow cytometry and droplet digital PCR (ddPCR) for TSDR analysis to assess the purity and stability of the cells, respectively. The regulatory T cell population was then cryopreserved.
  • ddPCR droplet digital PCR
  • the regulatory T cells were thawed and plated into 96 round bottom tissue-culture-treated plates at 50,000 cells per well in 200 ⁇ L X-VIVO 15 serum-free hematopoietic cell medium (Lonza, Cat#04-418Q).
  • the cells were split (1:2) every 3 to 4 days (Day 3, Day 6, Day 9, etc.) and activated with 25,000 T-activator CD3/CD28 DynabeadsTM. Each time the cells were split, the media was replaced with fresh X-VIVO 15 media supplemented with fresh CD3/CD28 DynabeadsTM and cytokines. At Day 0, Day 3, Day 6, and Day 9, FACS-based immunophenotyping was used to characterize the cells ( FIG. 2 ). At Day 0 (“Input”), cells were detected based on expression of FoxP3 and Helios expression. Cell expansion was also measured. At the endpoint, cells were collected for FACS analysis, and ddPCR was used to measure TSDR demethylation.
  • the data show that culturing regulatory T cells in the presence of IL-2 and at least one of the additional cytokines (IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, TNF- ⁇ , or the complete mixture) resulted in an improved level of regulatory T cell expansion, relative to a control, particularly when the concentration of the second cytokine was 10-1000 pg/mL ( FIG. 3 ).
  • the additional cytokines IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, TNF- ⁇ , or the complete mixture
  • the stability profile of the regulatory T cells was also assessed by determining the percentage of live cells that are CD4+ cells expressing the Treg marker FOXP3 (viable CD4 + Foxp3 + cells).
  • the data show that the regulatory T cells, in the presence of IL-2 and at least one additional cytokine from the mixture, at the concentrations shown, retain expression of FOXP3 at each of the cytokine concentrations tested, indicating that the cells stably retain a Treg phenotype in the presence of the cytokines ( FIG. 4 ).
  • the expansion and Treg purity were measured over a time-course ranging from 3-23 days in the presence of IL-2 and all 8 cytokines, each at the concentration shown.
  • the data show that the regulatory T cells continued to expand and remained stable for up to 23 days when cultured in the presence of the cytokine mixture of 1000 U/mL IL-2 and up to 1000 pg/mL of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ 1, and TNF- ⁇ ( FIGS. 5 A- 5 B ).
  • the fold expansion of the cell populations was measured on Days 4, 8, and 12. It was shown that the population of regulatory T cells grown in the presence of IL-2 and TNF- ⁇ was expanded by almost 120-fold over the period of twelve days ( FIG. 6 ). Conversely, the population of regulatory T cells grown in the presence of IL-2 alone was expanded by only 64-fold over the period of twelve days.
  • CD3/CD28 DynabeadsTM and IL-2 1000 U/mL
  • CD3/CD28 DynabeadsTM one of two different mixtures of pro-inflammatory cytokines: (1) IL-2 at 1000 U/mL and 1000 pg/mL of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , and TNF- ⁇ (Mix-6); or (2) IL-2 at 1000 U/mL and 1000 pg/mL of each of IL-1 ⁇ , IL-6, IL-12, IL-17A, IFN- ⁇ , TNF- ⁇ , IFN- ⁇ , and TGF- ⁇ (Mix-8).
  • the engineered regulatory T cells were exposed to control, Mix-6, or Mix-8 treatment over a period of 3 days.
  • FIGS. 7 A, 7 B and 7 D FACS analysis to determine FOXP3 expression and cell number and TSDR analysis before and after culture in the mixture of pro-inflammatory cytokines from three different donors showed that the regulatory T cells are stable after isolation, expansion, freeze-thaw and exposure to inflammatory cytokines ( FIGS. 7 A, 7 B and 7 D ).
  • FIG. 7 C is a repeat of the TSDR assay of FIG. 7 B done using an alternative bisulfite conversion method specifically designed for low cell counts.
  • FIG. 7 D Conventional T cells from the donor shown in FIG. 7 D were similarly cultured in the presence of IL-2 or one of the two different mixtures of pro-inflammatory cytokines (with CD3/CD28 DynabeadsTM in each instance). FACS and TSDR analysis showed that, in Mix-8, the conventional T cells are induced to express low levels of FOXP3, but TSDR stays hypomethylated, distinct from the stable regulatory T cells shown in FIG. 7 ( FIG. 8 A ).
  • FIG. 8 B is a repeat of the TSDR assay of FIG. 7 A done using an alternative bisulfite conversion method specifically designed for low cell counts.
  • FOXP3 expression can be induced in conventional CD4+ T cells in cell culture using TGF- ⁇ .
  • TGF- ⁇ TGF- ⁇
  • These T cells expressing high levels of FOXP3 induced by TGF- ⁇ are referred to as induced Tregs or iTregs and are capable of suppressing effector cell function.
  • iTregs are inherently unstable and removal of TGF- ⁇ can lead to the downregulation of FOXP3 and the reestablishment of their proinflammatory effector functions.
  • test material was made by sorting CD4 + T cells and dividing the cells into CD25 hi CD127 low Tregs (Tregs) and CD25 low Tconv cells.
  • T conventional cells were cultured with both IL-2 and TGF- ⁇ to induce iTregs for 7 days.
  • Tregs were cultured in the presence of IL-2 for only.
  • the cells were collected and washed to remove residual cytokine TGF- ⁇ and IL-2.
  • the cells were then placed in culture with either IL-2 alone or in the presence of IL-2 with Mix-6. On day 3, cells were fed with fresh media supplemented with IL-2.
  • FOXP3 protein expression FIG. 9 A
  • TSDR demethylation FIG. 9 B
  • a proportion of the cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 hours 5 days after the addition of inflammatory cytokines. This proportion of cells was then stained for the presence of intracellular IL-2 ( FIG. 9 C ) and IFN- ⁇ ( FIG. 9 D ) as readouts of effector cell function.
  • PMA phorbol 12-myristate 13-acetate
  • IFN- ⁇ FIG. 9 D
  • Tregs cultured in the presence of pro-inflammatory cytokines maintained low effector function as measured by IL-2 and IFN- ⁇ production. This data shows iTregs quickly lose their Treg phenotype, and this is observable within 5-days, while Tregs are stable, even in the presence of inflammatory cytokines. This supports the hypothesis that Treg instability is observed within the iTreg population, and this instability can emerge within 5 days of culture.
  • Tregs CD4 + CD25 + CD127 low regulatory T cells
  • Isolated Tregs were expanded for 12 days with lentiviral transduction to express a representative TCR.
  • Tregs were cultured with anti-CD3/CD28 antibody coated microbeads and recombinant human IL-2.
  • the engineered Tregs were then subjected to the method described in Example 1 to determine the stability profile of the engineered Tregs.
  • the engineered Tregs were cultured in the presence of (1) IL-2 alone, (2) IL-2 and a mixture of proinflammatory cytokines including IL-1 ⁇ , IL-6, IL-12 p70, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ , and TNF- ⁇ (Mix-8), or (3) IL-2 and a mixture of IL-1 ⁇ , IL-6, IL-12 p70, IL-17A, IFN- ⁇ , and IFN- ⁇ (Mix-6) for 5 days.
  • Non-lentiviral transduced but expanded CD4 + CD25 low CD127 high conventional T cells and CD4 + CD25 + CD127 low Tregs were used as control experiments using the same set of culture conditions.
  • the cell populations were cultured for a total of 5 days after a thaw.
  • Cytokines were added on the first day of culturing post-thaw (i.e., Day 0) and on Day 3.
  • TSDR demethylation, FOXP3 protein expression, and the production of intracellular proinflammatory cytokine IFN- ⁇ were measured on Day 0 and again on Day 5 (after activation with PMA and ionomycin for 4 hours).
  • Tregs (“TCR engineered Treg” populations) are phenotypically stable over 5 days when treated with proinflammatory cytokines, as TSDR demethylation status (>90% in total cell pellets ( FIG. 10 C ) and FOXP3 expression (>90% of CD4+ FOXP3+ cells among total live cells ( FIG. 11 C )) were maintained at Day 5, with minimal levels of IFN- ⁇ expression ( ⁇ 10% among total live cells ( FIG. 12 C )).
  • the untransduced Tregs (“Treg” populations) demonstrated similar phenotypic stability profiles as compared to the engineered Tregs ( FIGS. 10 B, 11 B, and 12 B ).
  • Tconv cells (“Tconv” populations) were not phenotypically stable with induced FOXP3 expression (up to 34.2%, FIG. 11 A ) and significantly upregulated IFN- ⁇ production (up to 32.0%, FIG. 12 A ), which suggests that the proinflammatory conditions used in the study exerted significant impact on the phenotype of the Tconv, and that this was observable in the 5-day duration of the assay. Additionally, Tconv cells had consistently low level TSDR demethylation ( ⁇ 7.0%, FIG. 10 A ) even in the conditions that provided induced expression of FOXP3 and IFN- ⁇ , indicating that the TSDR demethylation shown in Tregs and engineered Tregs did not result from induction of FOXP3.
  • regulatory T cells e.g., engineered regulatory T cells
  • inflammatory conditions such as inflammatory conditions that approximate the pro-inflammatory environment within a subject who has been administered the regulatory T cells
  • a period of time e.g., at least five days.
  • a cell population comprising regulatory T cells e.g., engineered regulatory T cells
  • regulatory T cells e.g., engineered regulatory T cells
  • two or more pro-inflammatory cytokines e.g., two or more cytokines selected from IL-2, IL-1 ⁇ , IL-6, IL-12 p70, IL-17A, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ , and TNF- ⁇
  • characterizing markers of stability of the cells e.g., FOXP3 expression, TSDR demethylation, and IFN- ⁇ production.

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